Approximately 40 unique secreted ligands, divided into three subgroups (TGFs, activins and BMPs) form the TGF-superfamily whose function is to coordinate numerous cellular events. TGF-family ligands are already being targeted therapeutically by blocking their interactions with receptors to alleviate human disorders such as cancer, fibrosis and muscle wasting disease. Naturally, ligands are regulated by either formation of an inactive covalent complex with their N-terminal propeptide that is cleaved during synthesis or by neutralization by one of a number of structurally-diverse extracellular antagonists. Binding of these molecules to ligands can range from very broad, where multiple ligands are antagonized by a single antagonist or very specific where one antagonist only recognizes a distinct ligand. While TGF-family ligands are structurally similar, the molecular basis of how structurally-diverse antagonists selectively neutralize subsets of ligands remains unclear. Therefore, our long-term goal of this application is to elucidate mechanisms of specificity among the TGF-family antagonists. This proposal will focus on the divergent ligand specificity among three activin-family antagonists: Follistatin (FST) (broad ligand specificity), Follistatin-like 3 (FSTL3) (moderate specificity), and propeptides (exquisite specificity). Our central hypothesis is that antagonists differentially interact with conserved and nonconserved ligand surfaces to confer a range of specificity within the activin-family of ligands. In Aim 1, we will determine how FST antagonists confer broad ligand antagonism through a combination of X-ray structure analysis of FST:ligand complexes and binding studies. In Aim 2, we will determine how FSTL3 domain differences restrict binding to a few ligands, and in Aim 3, we will establish how the propeptide forms an inactive complex with the myostatin ligand. In all three aims, we will combine X-ray structure determination with competition and surface plasmon resonance binding experiments along with cellular-based assays to elucidate mechanisms of specificity for each antagonist. Elucidating details of the mechanisms that neutralize TGF-family ligands will help in development of new strategies and optimize current therapies aimed at antagonizing ligands. PUBLIC HEALTH RELEVANCE Presently, TGF-family ligands are being neutralized therapeutically to alleviate human disorders such as cancer, fibrosis and muscle wasting. Our research is significant as it is expected to provide details of binding molecules that antagonize ligands. This information will strengthen efforts to customize therapies designed to neutralize TGF-family ligands.